The deregulation of the Sonic Hedgehog signaling pathway has been implicated in a variety of human cancers. Signaling through this pathway involves the activation of a seven transmembrane protein, Smoothened. This activation triggers the translocation of the transcription factor, Gli, to the nucleus where it regulates a variety of genes involved in cell growth, proliferation, and differentiation. The mechanisms of how Smoothened transmits the signal to the nucleus are unknown. The similarity of Smoothened to prototypical G-protein coupled receptors (GPCRs) may indicate that Smoothened can associate with proteins that are involved in GPCR signaling. Two such families of proteins are the G-protein coupled receptor kinases (GRKs) and the Beta-arrestin proteins. We propose to examine the role of GRK and beta-arrestin on Smoothened function by 1) characterizing the recently observed co-localization between beta-arrestin and Smoothened; 2) examining the importance of GRK/Beta-arrestin in Smoothened mediated signaling; and 3) by identifying any abnormal interactions between GRK/Beta-arrestin and the previously identified oncogenic Smoothened mutants, M1 and M2. These experiments aim to identify GRK and beta-arrestin as new components of the Smoothened signaling pathway. As this pathway is deregulated in several human cancers, proteins identified here as mediators of Smoothened signaling represent new targets for the development of therapeutic drugs that inhibit the pathway.